Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/µg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

Inhibition mechanism of trypsin by Schiff base metal chelate inhibitors.

Studies on trypsin-specific compounds are useful for the design of clinically useful compounds. It is well known that several benzamidine derivatives are potent competitive inhibitors of trypsin and trypsin-like enzymes. Many kinds of Schiff base metal chelate containing either amidine or guanidine have been synthesized and their inhibitory activities against trypsin have been characterized. Recently, the interactions of the Schiff base metal chelate inhibitors with trypsin enzyme have been determined by X-ray crystal structure analysis. The structural information and inhibitory activity data for amidine- and guanidine-containig Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.

Identification of a novel carbohydrate-mimicking octapeptide from chemical peptide library and characterization as selectin inhibitor.

We found a novel octapeptide (H-YRNWFGRW-NH2) mimicking sialyl Lewis X (sLe(X)) carbohydrate from a chemical peptide library with anti-sLe(X) monoclonal antibody (MAb) 2H5. The peptide libraries were constructed by Fmoc-based solid-phase methodology using the mix-split method. The octapeptide sequence was determined by the iterative deconvolution method using anti-sLe(X) MAb 2H5. To define the important residues for interaction with anti-sLe(X) MAb 2H5, alanine-scanning analogues of H-YRNWFGRW-NH2 were synthesized. Substitution of Tyr¹, Trp4, Arg7 and Trp8 to Ala resulted in a marked drop in affinity. This result indicates that aromatic and cationic amino residues have a key role in interacting with anti-sLe(X) MAb 2H5. The binding property of the octapeptide was evaluated with anti-sLe(X) MAb 2H5 and human E-selectin. The octapeptide showed high inhibitory potency (IC50=17.8 nM) for sLe(X) and competitively inhibited the binding of anti-sLe(X) MAb 2H5 in a dose-dependent manner. The octapeptide had high affinity (K(d)=0.168 µM) for E-selectin and this binding was inhibited by sLe(X). These results suggest that octapeptide binds to anti-sLe(X) MAb 2H5 or E-selectin at the sLe(X) binding site and sterically interferes with the recognition of anti-sLe(X) MAb 2H5 or E-selectin with sLe(X). This peptide may be a useful lead compound for an anti-inflammatory agent targeting selectin.

Structural basis for the design of novel Schiff base metal chelate inhibitors of trypsin.

The crystal structures of the complexes of bovine trypsin with m-guanidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 1), [N,N'-bis(m-guanidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 2), and [N,N'-bis(m-amidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 4) have been determined. The guanidine-containing trypsin-inhibitors (1 and 2) bind to the trypsin active site in a manner similar to that previously reported for amidine-containing inhibitors, for example, m-amidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 3). However, the binding mode of the guanidino groups of inhibitors 1 and 2 to Asp189 in the S1 pocket of trypsin was found to be markedly different from that of the amidino group of inhibitor 3. The present X-ray analyses revealed that the interactions of the metal ion of the inhibitors with the active site residues of trypsin play a crucial role in the binding affinity to the trypsin molecule. These structural information and inhibitory activity data for amidine- and guanidine-containing Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.

A structural comparison of three isoforms of anionic trypsin from chum salmon (Oncorhynchus keta).

Three anionic salmon trypsin isoforms (CST-1, CST-2 and CST-3) were isolated from the pyloric caeca of chum salmon (Oncorhynchus keta). The order of catalytic efficiency (K(m)/k(cat)) of the isoforms during BAPA hydrolysis was CST-2 > CST-1 > CST-3. In order to find a structural rationalization for the observed difference in catalytic efficiency, the X-ray crystallographic structures of the three isoforms were compared in detail. Some structural differences were observed in the C-terminal alpha-helix, interdomain loop and active-site region. From the results of the detailed comparison, it appears that the structural flexibility of the C-terminal alpha-helix, which interacts with the N-terminal domain, and the substrate-binding pocket in CST-3 are lower than those in CST-1 and CST-2. In addition, the conformation of the catalytic triad (His57, Asp102 and Ser195) differs among the three isoforms. The imidazole N atom of His57 in CST-1 and CST-2 forms a hydrogen bond to the hydroxyl O atom of Ser195, but the distance between the imidazole N atom of His57 and the hydroxyl O atom of Ser195 in CST-3 is too great (3.8 A) for the formation of a hydrogen bond. Thus, the nucleophilicity of the hydroxyl group of Ser195 in CST-3 is weaker than that in CST-1 or CST-2. Furthermore, the electrostatic potential of the substrate-binding pocket in CST-2 is markedly lower than those in CST-1 and CST-3 owing to the negative charges of Asp150, Asp153 and Glu221B that arise from the long-range effect. These results may explain the higher catalytic efficiency of CST-2 compared with CST-1 and CST-3.

Cloning, expression, purification and preliminary crystallographic studies of the adenylate/uridylate-rich element-binding protein HuR complexed with its target RNA.

Adenylate/uridylate-rich elements (AREs), which are found in the 3'-untranslated region (UTR) of many mRNAs, influence the stability of cytoplasmic mRNA. HuR (human antigen R) binds to AREs and regulates various genes. In order to reveal the RNA-recognition mechanism of HuR protein, an RNA-binding region of human HuR containing two N-terminal RNA-recognition motif domains bound to an 11-base RNA fragment has been crystallized. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 42.4, b = 44.9, c = 91.1 A. X-ray diffraction data were collected to 1.8 A resolution.

Crystallization and preliminary x-ray studies of the unliganded wild-type bovine thrombin.

Wild type of bovine thrombin has been crystallized in a ligand-free form by the hanging drop vapor diffusion method with polyethylene glycol 4000 and 2-propanol. The crystals belong to space group P4 3 2 12 with unit cell parameters of a = b = 87.7 A, c = 195.9 A. X-ray diffraction data were collected to 2.8 A resolution.

Kinetic properties of three isoforms of trypsin isolated from the pyloric caeca of chum salmon (Oncorhynchus keta).

Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K(m) values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 degrees C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k(cat), of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k(cat)/K(m)) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K(m) and k(cat) values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K(m) values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k(cat) values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k(cat)/K(m)) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k(cat)/K(m) values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k(cat)/K(m) values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

DNA recognition mechanism of the ONECUT homeodomain of transcription factor HNF-6.

Hepatocyte nuclear factor-6 (HNF-6), a liver-enriched transcription factor, controls the development of various tissues, such as the pancreas and liver, and regulates the expression of several hepatic genes. This protein belongs to the ONECUT class of homeodomain proteins and contains a bipartite DNA-binding domain composed of a single cut domain and a characteristic homeodomain. This transcription factor has two distinct modes of DNA binding and transcriptional activation that use different coactivators depending on the target gene. The crystal structure of the bipartite DNA-binding domain of HNF-6alpha complexed with the HNF-6-binding site of the TTR promoter revealed the DNA recognition mechanism of this protein. Comparing our structure with the DNA-free structure of HNF-6 or the structure of Oct-1, we discuss characteristic features associated with DNA binding and the structural basis for the dual mode of action of this protein, and we suggest a strategy for variability of transcriptional activation of the target gene.

Crystallization and preliminary X-ray studies of the DNA-binding domain of hepatocyte nuclear factor-6alpha complexed with DNA.

Hepatocyte nuclear factor 6 (HNF-6)/OC-1, a part of liver-enriched transcription factor, controls pancreas and liver development and regulates expression of several hepatic genes. DNA-binding region of HNF-6alpha bound to a 14-mer DNA fragment has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P2 with unit cell parameters of a = 73.0 A, b = 39.0 A, c = 106.5 A, beta = 107.6 degrees. X-ray diffraction data were collected to 2.0 A resolution.

Structure of the catalytic nucleotide-binding subunit A of A-type ATP synthase from Pyrococcus horikoshii reveals a novel domain related to the peripheral stalk.

H(+)-transporting ATP synthase is a multi-subunit enzyme involved in the production of ATP, which is an essential molecule for living organisms as a source of energy. Archaeal A-type ATPase (A-ATPase) is thought to act as a functional ATP synthase in archaea and is thought to have chimeric properties of F-ATPase and V-ATPase. Previous structural studies of F-ATPase indicated that the major nucleotide-binding subunits alpha and beta consist of three domains. The catalytic nucleotide-binding subunit A of V/A-ATPase contains an insertion of about 90 residues which is absent from the F(1)-ATPase beta subunit. Here, the first X-ray structure of the catalytic nucleotide-binding subunit A of an A(1)-ATPase is described, determined at 2.55 A resolution. A(1)-ATPase subunit A from Pyrococcus horikoshii consists of four domains. A novel domain, including part of the insertion, corresponds to the 'knob-like structure' observed in electron microscopy of A(1)-ATPase. Based on the structure, it is highly likely that this inserted domain is related to the peripheral stalk common to the A- and V-ATPases. The arrangement of this inserted domain suggests that this region plays an important role in A-ATPase as well as in V-ATPase.

Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.

Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos.

Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations. Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system. For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value. The value of epsilon;(c) increased with an increase in HU concentration. Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex. The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations. Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU.